1. Field of the Invention
The present invention relates to a method for determining the base sequence of nucleic acids, and an apparatus for said methods.
2. Background Art
Determining and specifying the base sequence of nucleic acids, particularly DNAs (deoxyribonucleic acids) and RNAs (ribonucleic acids), are important for elucidating genetic information and biological information of animals (human beings), plants, bacteria, viruses and the like.
Representative conventional methods for determining the base sequence of nucleic acids include a method, called "Sanger method" or "chain termination method," developed by F. Sanger and A. R. Coulson, and a method, called "Maxam-Gilbert method" or "chemical cleavage method," developed by A. Maxam and W. Gilbert.
The Sanger method will be briefly explained. A DNA fragment to be sequenced is first introduced into a single stranded DNA phage (for example, M13phage) and cloned. This recombinant DNA is used as a template to anneal a primer DNA, and a complementary oligonucleotide fragment is synthesized using a DNA polymerase. In this case, four deoxynucleotide phosphates as substrates and dideoxynucleotide phosphate as a reaction terminator are added to four separate reaction systems. Upon incorporation of dideoxynucleotide into oligonucleotide being synthesized, the synthesis of the oligonucleotide is terminated. As a result, various lengths of DNA fragments ending with dideoxynucleotide are synthesized. The DNA fragments are electrophoresed on a polyacrylamide gel to detect the fragments based on the label possessed by the DNA fragments, thereby determining the base sequence of the target DNA.
Next, the Maxam-Gilbert sequencing method will be briefly explained. The end of DNA to be sequenced is first labeled, and specific one or two bases are chemically cleaved. In this case, reaction conditions are set so that only several sites are nicked in any DNA. Treatment with piperidine permits DNA to be cut at the cleaved base site to provide various lengths of labeled fragments and unlabeled fragments. Only labeled fragments are involved in the sequencing. The larger the distance of the cleaved base from the labeled one end, the larger the size of the fragment. Electrophoresis permits the distance of the labeled one end to the cleaved base to vary according to the electrophoretic mobility. In this method, it is common practice to carry out the above chemical reaction so that four chemical reactions are carried out respectively to cleave only G, only G and A, only T and C, and only C.
On the other hand, in recent years, an attempt has been made to analyze genomes in a large number of organism species including human beings. In this attempt, there is a strong demand for processing of a large quantity of gene information in a short time. This has led to several proposals on the modification of conventional methods to process a large amount of gene information in a short time. For example, multi-capillary electrophoresis using a plurality of capillary columns has been proposed and put to practical use for processing of a large quantity of gene information in a short time. This method is advantageous in that a plurality of analytes can be analyzed. In this method, however, when one analyte is analyzed, four capillaries should be provided respectively for the bases. In order to simultaneously analyze a plurality of analytes, the number of capillaries should be further increased for each analyte. On the other hand, analysis of one analyte has been carried out using one capillary with a different label being used for each base. In this method, however, for simultaneous analysis of a plurality of analytes, the number of capillaries should be simply increased. Most of multi-capillary electrophoresing devices are constructed so that a plurality of capillaries are scanned by a laser beam or the like to read labels. Therefore, increasing the number of capillaries results in increased width to be scanned, leading to a fear of the detection accuracy being lowered.
Therefore, the development of a method for simultaneously analyzing a plurality of analytes in an efficient and accurate manner has still been desired in the art.
So far as the present inventor knows, there is no report that, in simultaneously analyzing a plurality of analytes by sequencing based on the Sanger method or the Maxam-Gilbert method, oligonucleotide fragments are simultaneously analyzed with a different label being used for each analyte.